Understanding gene expression and the relationship between genes, gene expression and disease is a fundamental goal of modern medicine. Gene expression is central to many forms of disease, including inherited diseases, infectious diseases, and cancer. Procedures for studying gene expression ultimately often rely on expression of genes in vivo, as do most gene therapy approaches.
Many procedures for achieving in vivo expression of genes have relied on transfection of cells with viral vectors such as adenoviral vector mediated gene delivery, e.g., to treat cancer (see, e.g., Chen et al. (1994) Proc. Nat'l. Acad. Sci. USA 91: 3054-3057; Tong et al. (1996) Gynecol. Oncol. 61: 175-179; Clayman et al. (1995) Cancer Res. 5: 1-6; O'Malley et al. (1995) Cancer Res. 55: 1080-1085; Hwang et al. (1995) Am. J. Respir. Cell Mol. Biol. 13: 7-16; Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 (Pt. 3): 297-306; Addison et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 8522-8526; Colak et al. (1995) Brain Res. 691: 76-82; Crystal (1995) Science 270: 404-410; Elshami et al. (1996) Human Gene Ther. 7: 141-148; Vincent et al. (1996) J. Neurosurg. 85: 648-654). Replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome have also been used, particularly with regard to simple MuLV vectors. See, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)).
The transfection of cells in vivo with nucleic acids complexed with lipids, rather than viral vectors, is also becoming increasingly useful as a tool for studying gene regulation in vivo and as a delivery method for gene therapy. Lipid-DNA complexes have been used to transfect cells with a variety of nucleic acids in a variety of mammals, including mice, rats, sheep, rabbits and humans. For example, Stribling et al. (1992) PNAS 89:11277-11281 describe transfection of murine lung cells with various reporter constructs delivered by aerosolization of lipid-DNA complexes. See also, Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662. Alton et al. (1993) Nature Genetics 5:135-142 describe transfection of mouse lung, trachea and intestine with DNA-lipid complexes which include cationic lipids and neutral lipids. The DNA encoded the gene for human CFTR (under the control of the commonly used CMV promoter). See also, McLachlan et al. (1995) Gene Therapy 2:614-622.
Canonico et al. (1994) AM. J. Respir. Cell Mol. Biol. 10:24-29 and Canonico et al. (1994) The Amencan Physiological Society 415-419 describe transformation e.g., of Rabbit lung and liver by delivery of DNA-lipid complexes comprising cationic lipids. Capelen et al. (1995) Nature Medicine 1(1):39 describe delivery and functional replacement of CFTR activity in the nasal epithelia of human patients having cystic fibrosis using cationic lipid-cholesterol: DNA complexes. Similarly, McLachlan et al. (1996) Gene Ther. 3(12): 1113-23 provided similar results using DNA-cationic lipid complexes.
Applications where gene therapeutic approaches are most helpful include those in which conventional treatments are inadequate. For example, peritoneal dissemination is one of the most common complications of malignancies of the digestive system, such as gastric or pancreatic cancers. Gene therapy for peritoneal dissemination of pancreatic cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase (a suicide gene) was performed in a nude mouse pancreatic cancer model. See, Aoki et al. (1997) Human Gene Therapy 8:1105-1113. Protection against peritoneal dissemination was observed in the model. Safety studies of the intraperitoneal injection of E1A-liposome complexes in mice have also been performed. The adenovirus 5 E1A gene has been reported to inhibit HER-2/ neu transcription and functions as a tumor suppressor gene in HER-2/ neu overexpressing cancer cells. Liposomal delivery of E1A prolongs survival of tumor-bearing mice. See, Xing et al. (1997) Gene Therapy 4:238-234.
A wide variety of DNA:lipid formulations have been demonstrated to be applicable to in vivo gene delivery and a very broad array of lipids have shown to have efficacy in at least one system. For example, Aoki, id. used dioctadecylamidoglycylspermine (DOGS):DNA complexes for in vivo transfection. Alton et al. (1993) Nature Genetics 5:135-142 used the cationic lipid (N-[1-(2,3- Dioleoyloxy) propyl]-N,N,N-trimethyl-amrnmoniummethyl-sulphate (DOTAP) for aerosol delivery of nucleic acids. Felgner, et al., (1987) Proc. Nat. Acad. Sciences, (USA) 84:7413-7417 describe the synthesis and use of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-triethylammonium chloride (DOTMA) for transfecting cells; the composition has been used for gene delivery (sold under the trade name Lipofectin.TM.). 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) synthesis is described in Stamatatos, et al., Biochemistry, (1988) 27:3917-3925; this lipid has also been used for in vivo gene delivery. DOTMA, DOTAP, Dimethyl dioctadecyl ammonium bromide (DDAB), or L-lysinyl-phosphatidylethanolamine (L-PE) and a second neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE) or cholesterol (Chol), have shown to be of use in in vivo cell transformation. See e.g., Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662. Heath (U.S. Pat. No. 5,698,721) describes cationic ampiphiles that are alkyl or alkyloxy-alkyl O-phosphate esters of diacylphosphatidyl zwitterionic compounds such as phosphatidylcholine or phosphatidylethanolamine and their use in transfecting cells with nucleic acids using the lipids as carrier molecules. Gorman WO 96/40963 describes the synthesis and use of O-ethyl-dimyristoylphosphatidylcholine (EDMPC) in conjunction with dioleoylphosphatidylethanolamine (DOPE) or cholesterol for gene delivery applications.
While lipid carriers have been shown to enhance nucleic acid delivery in vitro and in vivo, the mechanism by which they facilitate transfection is not clearly understood. While it was initially believed that lipid carriers mediated transfection by promoting fusion with plasma membranes, allowing delivery of the DNA complex into the cytoplasm, it is now generally accepted that the primary mechanism of cellular uptake is by endocytosis. While the mechanism by which cationic lipid carriers act to mediate transfection is not clearly understood, they are postulated to act in a number of ways with respect to both cellular uptake and intracellular trafficking. Some of the proposed mechanisms by which cationic lipids enhance transfection include: (i) compacting the DNA, protecting it from nuclease degradation and enhancing receptor-mediated uptake, (ii) improving association with negatively-charged cellular membranes by giving the complexes a positive charge, (iii) promoting fusion with endosomal membranes facilitating the release of complexes from endosomal compartments, and (iv) enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed. When used for in vivo delivery, the role of the cationic lipid carriers is further complicated by the interactions between the lipid-nucleic acid complexes and host factors, e.g., the effects of the lipids on binding of blood proteins, clearance and/or destabilization of the complexes.
Typically, cationic lipids are mixed with a non-cationic lipid, usually a neutral lipid, and allowed to form stable liposomes, which liposomes are then mixed with the nucleic acid to be delivered. The liposomes may be large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). The liposomes are mixed with nucleic acid in solution, at concentrations and ratios optimized for the target cells to be transfected, to form cationic lipid-nucleic acid transfection complexes. Alterations in the lipid formulation and mode of delivery allow preferential delivery of nucleic acids to particular tissues in vivo. See, PCT patent application Nos. WO 96/40962, WO 96/40963.
Thus, one problem in the art is the difficulty in identifying relevant parameters for lipid-mediated nucleic acid delivery. An additional problem is that there are so many liposomal formulations available, that it is difficult to test all possible formulations in vivo for a particular application. The present invention overcomes these problems, providing in vitro assays for selecting liposomal formulations for in vivo delivery, parameters which are important for in vivo delivery and, importantly, particularly desirable liposomal formulations for particular applications such as transfection of tumor cells in the peritoneal cavity.